Hermansky-Pudlak syndrome (HPS) is a group of related genetic disorders that result in oculocutaneous albinism (OCA), bleeding disorders and often lethal lung fibroses. These symptoms are due to defects in the biogenesis and function of cell type-specific lysosome related organelles (LROs) in affected cell types, such as melanosomes (the organelles in skin and eye pigment cells in which melanin pigments are synthesized and stored), platelet dense granules and lung epithelial cell lamellar bodies. Notably, individuals with OCA suffer from poor visual acuity and are at significantly increased risk of skin cancer due to loss of UV protection by melanin and to retinal degeneration. Five of the eight subtypes of HPS in humans result from mutations in two protein complexes, Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 and BLOC-2. The molecular function of these complexes is not known, and understanding of their mechanistic role in LRO biogenesis will likely lead to novel therapeutic approaches for HPS. This proposal will investigate the specific roles of BLOC-1 and BLOC-2 in protein transport using melanosomes as model LROs and immortalized melanocyte cell lines from wild type and HPS model mice as an experimental system. I hypothesize that BLOC-1 and BLOC-2 function in conjunction with the endosomal SNARE protein, syntaxin13 (STX13), and other partner SNARE proteins to regulate the dynamics of recycling endosome-derived transport intermediates and to specify their docking and fusion with melanosomes. The specific aims of this proposal are: (1) to test whether BLOC-2 regulates anterograde cargo delivery specifically to melanosomes~ (2) to test whether BLOC-1 and BLOC-2 regulate the dynamics of endosomal transport intermediates by directing them to contact melanosomes~ and (3) to test whether BLOC-1 and BLOC-2 influence the composition of STX13-containing SNARE complexes in melanocytes. To achieve these aims, I will compare the behavior of melanosome and endosomal cargoes in wild-type, BLOC-1- and BLOC-2-deficient melanocytes by: quantitative live cell microscopy to investigate interactions of STX13-containing endosomal carriers with melanosomes~ flow cytometric analyses of endocytic dynamics to assess melanosome cargo trafficking~ and immunofluorescence and immunoelectron microscopy to assess steady state distribution of melanosome cargoes. I will exploit in vitro and ex vivo techniques for assessing protein-protein interactions and mass spectrometry to identify STX13 binding partners. These studies will provide insights into the molecular basis for LRO biogenesis and clarify the specific events which segregate LRO cargoes from the endosomal system and allow for maturation of specialized LROs. This work will provide insight into the molecular mechanisms of HPS, identify potential therapeutic targets for treatment of HPS and possibly other types of albinism and increase our understanding of diseases affecting LROs.